Differential Phosphorylation of Two Size Forms of the Neuronal Class ...

Vol. 268, No.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Issue of September 15,pp. 19461-19457,1993 Printed in U.S.A.

Differential Phosphorylation of Two Size Forms of the NeuronalClass C L-type Calcium Channel a1 Subunit* (Received for publication, April 12, 1993, and in revised form, May 1, 1993)

Johannes W. Hell, Charles T. YokoyamaS, Scott T. Wong, ConcepcionWarner, TerryP. SnutchBV, and William A. Catterall From the Department of Pharmacology and $Graduate Program in Neurobiology, University of Washington, Seattle, Washington 98195 and the §Biotechnology Laboratory and Departments of Zoology and Neuroscience, University of British Columbia, Vancouver, British Columbia V6T 1 W5, Canada

L-type calcium channels mediate long-lasting calcium currents which are modulated by proteinphosphorylation. Using site-directed anti-peptide antibodies, we show that the a1 subunit of the neuronal class C L-typecalcium channel from rat brain exists in two size forms. The longer form, Lcz, with an apparent molecular mass of 210-236 kDa was phosphorylated in vitro by CAMP-dependent protein kinase (cA-PK), but the shorter form,LCl, with an apparentmolecular mass of 190-196 kDa was not a substrate for cA-PK. In contrast,LCl and Lcz are both substrates for protein kinase C (PKC), calcium- and calmodulin-dependent protein kinase11, and cGMP-dependent protein kinase (cG-PK). The site-directed anti-peptide antibody CNC2 was produced against the COOH-terminal end of the class C L-type a1 subunit as predicted by molecular cloning and sequencing of cDNA. CNC2 recognized Lca but not LCl by immunoblotting and immunoprecipitated only LCZphosphorylated by either cA-PK or PKC. These results indicate that LCl is truncatedat its COOH-terminal end with respect to LCZand that cA-PK preferentiallyphosphorylatessitesin the COOH-terminal region of the a1 subunit that arepresent in Lcz but not LCI. The selectivity of cA-PK for phosphorylation of the COOH-terminal region of Lcz suggests that the channel activities of the two a1 subunit size forms may be differentially regulatedby neurotransmitters and hormones which act through CAMP-dependent mechanisms, while both a1 subunit isoforms may be modulated by PKC, cG-PK, and calcium- and calmodulin-dependent protein kinase 11.

Calcium influx through voltage-dependent calcium channels controls a variety of neuronal functions including integration of electrical signals and release of neurotransmitters. Four types of voltage-sensitive calcium channels, designated T, L, N, and P, have been defined by electrophysiological and pharmacological criteria (Bean,1989; Llinas et al., 1989; Hess, 1990). L-type channels mediate long-lasting calcium currents and are blocked by dihydropyridines and other organic cal-

* This work was supported by National Institutes of Health Research Grants Pol-NS20482 (Philip Schwartzkroin, PrincipalInvestigator) and R01-NS22625 (to W. A. C.), by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to J. W. H.), and by Miles Laboratories and the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. ll Recipient of a research fellowship from the Alfred P. Sloane Research Foundation.

cium channel blockers (see Glossmann and Striessnig (1990) for review). L-type channels from skeletal muscle consist of five subunits: a l , a2, p, y, and 6 (Takahashi et al., 1987; Campbell et al., 1988; Catterall et al., 1988). cDNAs corresponding to all subunits have been cloned and sequenced (Tanabe et al., 1987; Ellis et al., 1988; Ruth et al., 1989; Jay et al., 1990). The a 2 and 6 subunits are encoded by the same gene and created by post-translational processing (De Jongh et al., 1990; Jay et al., 1991). a1 is the pore-forming subunit (Perez-Reyes et al., 1989; Mikami et al., 1989), but co-expression of other subunits affects its functional properties (Wei et al., 1991;Varadi et al., 1991; Singer et al., 1991). a1 subunits are phosphorylated by CAMP-dependent protein kinase (cAPK)’ andprotein kinase C (PKC) (Curtis and Catterall, 1985; Flockerzi et al., 1986; Takahashi et al., 1987; Nastainczyk et al., 1987; O’Callahan and Hosey, 1988). Phosphorylation by cA-PK enhances the activation of purified skeletal muscle calcium channels reconstituted in planar phospholipid bilayers or phospholipid vesicles (Flockerzi et al., 1986; Hymel et al., 1988; Nunoki et al., 1989; Mundiiia-Weilenmann et al., 1991). Two size forms have been detected for skeletal muscle calcium channel a1 subunits, a full-length form and a form that is truncated at the carboxyl-terminal (De Jongh et al., 1989,1991). These two forms of the calcium channelare differentially phosphorylated by cA-PK in vitro and in intact skeletal muscle cells (Lai et al., 1990; Rotman et al., 1992). The subunit composition of neuronal L-type channels is similar to that of skeletal muscle L-type channels including subunits resembling the a1, a2,p, and 6 polypeptides (Takahashi and Catterall, 1987; Ahlijanian et al., 1990). Based on the recent cloning and sequencing of cDNAs encoding five different classes of calcium channel a1 subunits from mammalian brain (Snutch et al., 1990; Hui et al., 1991; Snutch et al., 1991; Williams et al., 1992a,1992b; Dubel et al., 1992; Seino et al., 1992; Niidome et al., 1992), we produced antibodies against peptides corresponding to sequences specific to each of these channels (Dubel et al., 1992; Westenbroek et al., 1992). The antibody CNCl is directed against a central intracellular domain of the rat brain class C L-type a1 subunit. The CNCl antibody selectively immunoprecipitates radiolabeled dihydropyridine-binding sites from rat brain (Dubel et al., 1992), and the class C a1 subunit directs the functional expression of L-type calcium channels.* Although neuronal L-type calcium channels are regulated by neurotransmitters ‘The abbreviations used are: cA-PK, CAMP dependent protein kinase; BSA, bovine serum albumin; CaM kinase 11, calcium- and calmodulin-dependent protein kinase 11; PAGE, polyacrylamide gel electrophoresis; cG-PK, cGMP-dependent proteinkinase; PKC, protein kinase C; PAS, Protein A Sephsrose;TBS, Tris-buffered saline. J. Tomlinson, A. Stea, and T. Snutch, unpublished results.

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Phosphorylation of NeuronalChannels Calcium

and hormones acting through cA-PK and PKC (see Dolphin (1990) and Miller (1990), for review), phosphorylation of the protein components of these channels has not yet been described. In this report, we use site-directed anti-peptide antibodies which recognize different size forms of the class C a1 subunit toexamine their differential phosphorylation by second messenger-activated serinelthreonine protein kinases. EXPERIMENTAL PROCEDURES

M~terials-[~H]Isradipine (PN200-110;3200 GBq/mmol) and [y3'P]ATP (111TBq/mmol) were purchased from Du Pont-New England Nuclear, the ECL detection kitfor immunoblotting from Amersham Corp., digitonin from Gallard-Schlesinger (Carle Place, NY), and protein A-Sepharose (PAS) and heparin-agarose from Sigma. cA-PK and PKC were purified by standard procedures (Kaczmarek et al., 1980; Woodgett and Hunter, 1987) and kindly provided by Drs. E. I. Rotman and B. J. Murphy, Department of Pharmacology, University of Washington. Calcium- and calmodulin-dependent protein kinase I1 (CaM kinase 11) expressed in Sf9 cells using a baculovirus expression vector and purified to homogeneity was a generous gift of Drs. D. A. Brickey and T. Soderling, Vollum Institute, Portland, OR. cGMP-dependent protein kinase (cG-PK) was a generous gift from Dr. J. Corbin, Vanderbilt University, Nashville, TN and was also obtained commercially from Promega (Madison, WI). Control antibodies (rabbit IgG) were received from Zymed (South San Francisco, CA). All otherreagents were of standard biochemical quality from commercial sources. Two-month-old Sprague-Dawley rats were obtained from Bantin and Kingman (Bellevue, WA). Production and Purification of Peptides and Antibodies-CNC1 was produced against a highly variable site in the intracellular loop between domains I1 and 111 of the class C L-type a1 subunits (Dubel et al., 1992) and anti-CP(1382-1400) against the highly conserved amino acid sequence following the transmembrane segment IVS6 of the skeletal muscle and class C L-type a1 subunits (Striessnig et al., 1990). CNC2 was raised against the peptide KYGRGQSEEALPDSRSYVS corresponding to the predicted COOH-terminal amino acids 2122-2138 of the class C L-typea1 subunit (Snutch etal., 1991). The sequences that the CNC1, CNC2, and anti-CP(1382-1400) were raised against are completely conserved in all isoforms of the neuronal class C a1 subunit that aregenerated by alternative splicing (Snutch et al., 1991). A t the NH2 termini of the peptides, lysine and tyrosine residues were added to facilitate cross-linking and radiolabeling and are not part of the channel sequence. Peptides were synthesized, purified, coupled to bovine serum albumin, and used for immunization of rabbits, and the resulting antibodies were affinity-purified on peptide columns as detailed previously (Westenbroek et al., 1992). Immunoprecipitation and Phosphorylation of Partially Purified Ltype Calcium Channels-Neuronal calcium channels were solubilized from rat brain membranes with digitonin and enriched by wheatgerm agglutinin-Sepharose chromatography and sucrose gradient centrifugation as described previously (Westenbroek et al., 1992). One ml of a sucrose gradient fraction containing a t least 50 fmol of [3H] isradipine-labeled L-type calcium channels was preadsorbed with 150 pl of Sepharose CL-4B and 5 mg of PAS for 30 min. After centrifugation for 30s in a tabletop centrifuge, the supernatantwas collected and incubated on a tilting mixer for 1.5 h on ice with either of the following affinity-purified antibodies: CNC 1 (15 pg), CNC 2 (60 pg), or control antibody (60 pg). Then 2-3mgof PAS, preswollen and washed three times with phosphate-buffered saline, 0.5% BSA, 0.1% digitonin, were added, and the samples were mixed for 2.5 h in ice. The immune complexes were pelleted by centrifugation and washed three times with phosphate-buffered saline, 0.1% BSA, 0.1% digitonin and once with basic phosphorylation buffer (50 mM HEPES-NaOH, pH 7.4, 10 mM MgCl', 1 mM EGTA, 0.1% digitonin) supplemented with 0.1% BSA. The pellet was phosphorylated with 0.5-1 pg of cAPK, PKC, or CaM kinase I1 or with 0.1 pg of cG-PK in 50 pl of basic phosphorylation buffer containing 1 mM dithiothreitol, 1 pM pepstatin A, 2 pgof leupeptin, 4 pg of aprotinin, and 0.2 p~ [y3'P]ATP. This buffer was supplemented with 1.5 mM CaCI', 50 pg of diolein, and 2.5 mg of phosphatidylserine for PKC, 2 p~ cGMP for cG-PK, or 1mM calmodulin and 0.5 mM CaCI2without EGTA for CaM kinase 11. The samples were incubated for 30 min a t 32-34 "C, gently mixed every 2 min, washed twice with 0.1% digitonin in RIA buffer (25 mM Tris-HCI,pH 7.4,20 mM EDTA, 75 mM NaCl, 20 mM sodium pyrophosphate, 20 mM 8-glycerolphosphate, 50 mM NaF, 1 mM pnitrophenylphosphate), twice with 1%Triton X-100 in RIA buffer,

and once in 10 mM Tris-HCI, pH 7.4. The pellets were extracted for 30 min a t 50-60 'C with SDS sample buffer (20 p1 of 125 mM TrisHCI, pH 6.8, 2% SDS, 2 mM EDTA, 10% sucrose, 20 mM dithiothreitol, 1p~ pepstatin A, 2 pg/ml leupeptin, 4 pg/ml aprotinin, 1 pM p nitrophenylphosphate) and used directly for SDS-PAGE. For double immunoprecipitations, the pellets were treated with 30 pl of 50 mM Tris-CI, pH 7.4,1% SDS, 5mM dithiothreitol, 20 mM B-glycerolphosphate, 1 p~ p-nitrophenylphosphate, 1 p~ pepstatin A, 2 pgof leupeptin, and 4 pg of aprotinin for 30 min a t 50-60 'C, diluted with 300 pl of 1% TritonX-100, 0.1% BSA, 20 mM B-glycerolphosphate,1 p~ p-nitrophenylphosphate, 1p~ pepstatin A, 2 pg of leupeptin, and 4 pg of aprotinin in RIA buffer and centrifuged. The supernatantwas collected and incubated on ice with 25pgof affinity-purified antiCP(1382-1400) for 1.5 h. Two mgof PAS, pretreated as described above, were added and the samples incubated on a tilting mixer for 2.5h. ThePAS-anti-CP(1382-140O)d complexwas pelleted by centrifugation, washed three times with 1% Triton X-100 in RIA, and once with 10 mM Tris-HC1, pH 7.4, extracted with SDS sample buffer as before, and loaded onto an SDS-polyacrylamide gel. Zmrnunoblotting of Calcium Channels-Neuronal L-type calcium channels bind to heparin-agarose (Sakamoto and Campbell, 1991), which was used to concentrate the channels from sucrose gradient fractions. Forty pl of heparin-agarose were added to 1 mlof the channel fraction and incubated on a tilting mixer for 2-3 h at 4 "C. The resin was washed three times with 10 mM Tris-CI, pH 7.4, and 0.1% digitonin. The channels were extracted and immunoblotted as described previously (Westenbroek et al., 1992). For some experiments, polyacrylamide gelswere polymerized from 6% acrylamide instead of 576, as indicated in the figure legends. RESULTS

Phosphorylution of the Neuronal Class C L-type Calcium Channel by cA-PK and PKC-The antibody CNCl recognizes two size forms of the calcium channel a1 subunit in immunoblotting experiments(Fig. 1).The apparentmolecular mass of the larger polypeptide designated LC2, as determined by comparison with marker proteins, varied between 210 and 235 kDa depending on the concentration of acrylamide used for SDS-PAGE (Fig. 1). In a 5% acrylamide gel, the polypeptide migrated to a position just above myosin, the 205 kDa marker, whereas in a 6% acrylamide gel its position was close to the longer form of spectrin, the 240 kDa marker. The shorter form designated LCl migrated in both gels with an apparent molecular mass of190-195 kDa when compared with the marker proteins. Analogous observations were made during thedetermination of the molecular masses of the two size forms of the skeletal muscle L-type calcium channel a1 subunit (De Jongh et al., 1991). Although itcannot be excluded thatis created by in vitro proteolysis, this seems unlikely. Inclusion of high concentrations of several protease inhibitiors and careful pre5%

"

6%

FIG.1. Two size forms of the a1 subunit of the class C Ltype calcium channel. Neuronal calcium channels were partially purified from solubilized rat brain membranes by wheat germ agglutinin chromatography and sucrose gradient centrifugation, precipitated with heparin-agarose, and separated by SDS-PAGE using either 5 or 6% polyacrylamide gels as indicated at the top of the immunoblots. Immunoblotting was performed in both cases with affinitypurified CNC1. The molecular mass markers, indicated at the right side of each gel, were: a spectrin (240 kDa), 0 spectrin (220 kDa), myosin (205 kDa), and a2 macroglobulin (covalently linked to Coomassie Brilliant Blue, 191 kDa).

Phosphorylation ofChannels Calcium Neuronal

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cooling of all instruments including rotors to 0 “C did not than thea1 subunit itself. T o exclude other proteins from the alter the appearanceof Lcl (see Westenbroek et al. (1992), for immunoprecipitates, double immunoprecipitation experia detailed description). In addition, perfusion of rats with the ments were performed.After precipitation by CNCland membrane-permeable protease inhibitors leupetin and calpain phosphorylation by cA-PK or PKC, the protein A-antibodyinhibitors 1 and 2 to prevent post mortem proteolysis (Wes- calcium channel complex was washed and dissociated by tenbroek et al., 1992) did not reduce the relative amount of denaturation with SDS and dithiothreitol(see “Experimental LC, compared toLC2. Procedures”). This treatment was similar to that used to The antibody CNCl was used to selectively immunoprecip- dissociate the calcium channel complex for SDS-PAGE and itate the neuronal class C L-type calcium channel from a n therefore should completely dissociate the calcium channel enriched channel fraction. After incubation with cA-PK in subunits and associated proteins. A second immunoprecipithe presence of [-p3’P]ATP, only one band corresponding to tation was then performed with anti-CP(1382-1400). This the longer form of the class C L-type a1 subunit, Lc2, could antibody recognizes a segment of the a1 subunit whose sebe detected by autoradiography (Fig. 2, lanes 2 and 7). As quence isconserved in calcium channels so far characterized, observed in immunoblots for LC*,the phosphorylated band and it immunoprecipitates both L-type and N-type calcium migrated with an apparentmolecular mass of 210 kDa in 5% channels (Striessniget al., 1990; Ahlijanian et al., 1991). This acrylamide gels (Fig. 2, lane 2 ) or 235 kDa in 6% acrylamide sequence is accessible to anti-CP(1382-1400) only after reused. moval of the a2 andd subunits. Following the double immugels (Fig. 2, lane 7) as compared to the marker proteins This band was not observed if nonimmune antibodies were noprecipitation with CNClandanti-CP( 1382-1400), the used instead of CNCl (Fig. 2, lanes 1 and 6 ) demonstrating same phosphoprotein bands were detected by SDS-PAGE and the specificity of immunoprecipitation by CNC1. autoradiography as after thesingle immunoprecipitation. cAIn contrast, PKC phosphorylated both the LCl and Lc2 P K phosphorylated a polypeptide of 235 kDa, andPKC forms of the a1 subunit (Fig. 2, lane 4 ) . Both phosphoproteins phosphorylated twopolypeptides of 195and 235 kDa as were specifically precipitated by CNC1, since neitherof them resolved in 6% acrylamide gels (Fig. 3, lanes 1 and 3, respecwas detectable after immunoprecipitation with control antitively). No bands were observed if nonspecific antibodies were bodies (Fig. 2, lane 3). No phosphorylation was observed if used in the first immunoprecipitation(Fig. 3, lunes 2 and 4 ) . exogenous protein kinase was not added (Fig. 2, lane 5). These findings show conclusively that the phosphorylated Thestoichiometry of proteinphosphorylation was esti- polypeptides are a1 subunits and that only Lc2 was phosmated by comparison of the number of calcium channels in phorylated by cA-PK, although bothLcl and Lc2were present the immunoprecipitated samples based on specific binding of and phosphorylatedby PKC. the high affinity dihydropyridine antagonist isradipine Differential Recognition of the Two Size Forms of theClass (PN200-110) andtheincorporation of radiolabeled phos- C L-type a1 Subunit by an Antibody Directed Toward the , incor- COOH Terminus-As previously shown for the skeletal musphate. At limiting ATP concentration (0.2 p ~ )cA-PK porated 0.61 mol of phosphate/mol of Lc2 immunoprecipitated cle L-type a1 subunit (De Jongh et ul., 1989,1991), the shorter by CNC2(see below). At10 p~ ATP,theincorporation form of the neuronal class C L-type channel, Lcl, may be increased to 0.85 mol of phosphate/mol ofLc2. PKC incor- truncated at the COOH-terminal end in comparison to Lc2. porated 0.45 mol of phosphate/mol of Lcl plus Lc2 immuno- To test this hypothesis, the site-directed anti-peptide antiprecipitated by CNCl at0.2 p~ ATP and0.81 mol/mol a t 10 body CNC2 was produced against a peptide corresponding to pM ATP. the amino acid sequence of the COOH terminus as predicted Identification of the Phosphorylated Polypeptidesas a1 Sub- from cDNA sequenceof the class CL-type a1 subunit (Snutch units-L-type calcium channels are multisubunit complexes et al., 1991). Immunoblotting revealed that CNC2 recognized and may also interact with other cellular components such as only one form of the class C L-type a1 subunit, Lc2, with an cytoskeletal proteins and synaptic vesicle proteins. Therefore, apparent molecular mass of 235 kDa in the 6% gel (Fig. 4 A , it ispossible that the phosphorylated proteins immunopreciplane 2 ) . Lc2 and Lclwere identified on the same blot by CNC1, itated by the CNCl antibody under native conditions might which detected two proteins, the larger comigrating with the be associated proteins of similar size to thea1 subunit rather CNC2 antigen (Fig. 4A, lane 1). The specificity of the inter1 2 3 4 5 6 7 ~

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FIG. 2. Phosphorylation of the class C L-type calcium channel by cA-PK and PKC. Class C L-type calcium channels were immunoprecipitated with CNC1 (lanes 2,4,5, and 7 ) or with control antibodies (lanes I , 3,and 6 ) ,and phosphorylatedwith cA-PK (lanes I, 2, 6, and 7), PKC (lanes 3 and 4 ) , or no external protein kinase present (lane 5 ) . Polypeptides were separated by SDS-PAGE, using either a 5% polyacrylamide gel (lanes 1-5) or a 6% polyacrylamide gel (lanes 6 and 7). Molecular mass markers are indicated as in Fig. 1.

240 2 20 205 191

FIG.3. Identification of the class C L-type calcium channel phosphopeptides as a1 subunits by double immunoprecipitation. Double immunoprecipitation experiments were performed applying either CNCl (lanes 1 and 3 ) or control antibodies (lanes 2 and 4 ) for the first precipitation and anti-CP(1382-1400) for the second. Samples were phosphorylated by cA-PK (lanes 1 and 2 ) orPKC (lanes 3 and 4 ) and run on 6% polyacrylamide gels. Molecular mass markers are indicated as in Fig. 1.

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action of CNC2 with Lc2 was confirmed by competition experiments. Addition of 1 ~ L MCNC2 peptide to the CNC2 antibody solutionblocked the binding of this antibody toLC2 completely, and no immunoreactivitycould be detected even if the immunoblot was overexposed (Fig. 4A, lanes 3 and 4 ) . These results show that the twosize forms of the class C Ltype a1 subunits differ a t their COOH-terminal ends, Lcl being a truncated form of LCz. In order to determine the amount of Lc2 relative to the totalnumber of class C L-type calcium channels,L-type calcium channels were radiolabeled by binding of [3H]isradipine, solubilized with digitonin, partially purified, and immunoprecipitated with increasing concentrations of CNCl and CNC2. CNCl immunoprecipitated approximately 48% of the L-type calcium channels in this fraction at a saturating concentration (Fig. 5 A ) . In contrast, CNCB immunoprecipitated only 23% of the L-type calcium channels a t concentrationsapproachingsaturation.Preincubation of theCNC2

antibody with 1 or 10 p~ of the CNCB peptide reduced the immunoprecipitation of the L-type calcium channel by 80%, while preincubation with a control peptide derived from the class D L-type calcium channel (CND2) had no effect (Fig. 5 B ) . These results confirm thespecificity of the CNC2 antibody and suggest that approximately half of the class C Ltype calcium channels have a1 subunits which correspond to LC% Specific Immunoprecipitation and Phosphorylation of the Longer Form of the Class C L-type a1 Subunit"CNC2 was utilized for the immunoprecipitation andphosphorylation of class C L-type channels. Theimmunopurified channels were phosphorylated with cA-PK or PKC and reprecipitatedwith anti-CP(1382-1400). As expected,only the high molecular mass form of the class C a1 subunit with an apparent mass of 235 kDa was present in both cases (Fig. 6, lanes 3 and 5). This band corresponded to Lc2, since it comigrated with this form of the classC L-type a1 subunit as identified after precipitation with CNCl and phosphorylation by cA-PK (Fig. 1 2 3 4 6, lane 1). No phosphorylated proteins were detected when nonimmuneantibodies were used (Fig. 6, lanes 2 and 4 ) demonstrating the specificity of CNCZ. Virtuallyidentical results were obtained after single immunoprecipitations with CNCB (not shown). These experimentsconfirm that thephosY a: phorylated protein band comigrating with Lc2 is the longer bd -1 91 form of the classC L-type a1 subunit and that it contains the COOH terminus encoded by the cloned cDNA for the class C a1 subunit. Lcl, which is phosphorylated by PKC, was not FIG.4. Recognition of the two size forms of the class C L- immunoprecipitated by CNC2 providing additional evidence type a1 subunit by CNCP. Lanes I and 2, immunoblots were that Lcl and Lc2 differ at their COOH-terminal ends. performed as described in Fig. 1 with either CNCl (lane I ) or CNC2 Phosphorylation by CaM Kinase II and cG-PK-To deter(lane 2). CNCl detected both formsof the class C L-type 01 subunit, mine whether the classC L-type calcium channel a1 subunit LC, and Lc2, whereas CNC2 recognized only Lc2. Lanes 3 and 4, immunoblots were performed as in Fig. 1 with CNC2 (lane 3 ) or is also a substrate for phosphorylation by CaM kinase I1 and CNCB that had been preincubated overnight with1p~ CNC2 peptide. cG-PK, partiallypurified calcium channels were immunoprecipitated by CNC1, phosphorylated, and reimmunoprecipiMolecular mass markers are as indicated in Fig. 1.

A

B

CNCl Antibody [pg] 0

5

10

20

15

25

T

//CNC2

Concentration: 0

100

200

300

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Peptide:

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1pM 10pM CNC2 CNCZ CND2

IOpM

CNC2 Antibody [pg] FIG.5. Quantitative immunoprecipitation of the class C L-type calcium channel by C N C l and CNCZ. L-type calcium channels in rat brain membranes were radiolabeled by binding of [3H]isradipine and solubilized with digitonin as described previously (Westenbroek et al., 1992). A , the solubilized L-type calcium channels were immunoprecipitated with the indicated concentrations of CNCl (top abscissa) or CNCZ (lower abscissa) and quantitated by scintillation counting. B, 50 pgof CNCZ antibody were preincubated with the indicated concentrations of the CNC2 peptide or a control peptidederived from the amino acid sequence of the class D L-type calcium channel (CNDZ). CNC2 antibody was then used in immunoprecipitation assays as in panel A .

Phosphorylation of Neuronal Calcium Channels

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FIG.6. Specific immunoprecipitation of LC*by CNCS. Class C L-type a1 subunits were immunoprecipitated with CNCl (lane 1 ), CNC2 (lanes 3 and 5 ) , or control antibodies (lanes 2 and 4 ) , phosphorylated with either cA-PK (lanes 1-3) or PKC (lanes 4 and 5) and reprecipitated withanti-CP(1382-1400). Molecular mass markers are given as in Fig.1. 1

2

3

4

5

6

2402 20205191-

FIG.7. Phosphorylation of the classC L-type calcium channel a1 subunit byCaM kinase I1 and cG-PK. Class C L-type calcium channel a1 subunits were immunoprecipitated with CNCl (lanes I , 3, and 5 ) or with control antibody (lanes 2, 4, and 6) and phosphorylated with cG-PK (lanes 1 and 2 ) , CaM kinase I1 (lanes 3 and 4 ) , or PKC (lanes 5 and 6 ) and reprecipitated with anti-CP(13821400). Molecular mass markers are as described in Fig. 1. tated byanti-CP(1382-1400). CaMkinase I1 andcG-PK phosphorylated both the LCl and Lc2 like PKC (Fig. 7), in contrast to cA-PK which phosphorylated only Lc2 as shown above. Evidently, sites for phosphorylation by CaM kinase I1 and cG-PK are present in both size forms of the class C Ltype a1 subunits. DISCUSSION

The LCl and L e Size Formsof the Class C L-type a1 Subunit Differ at their COOH-terminal Ends-CNC1, a n antibody specific for the classC L-type a1 subunit, identified two size forms of this subunit by immunblotting and by immunoprecipitation and phosphorylationby PKC, CaM kinase 11, and cG-PK. Similar findings were recently obtained for neuronal class B N-type, cardiac L-type, and skeletal muscle L-type calcium channels (De Jongh et al., 1989, 1991; Yoshida et al., 1992; Westenbroeket al., 1992). Fortheskeletal muscle calcium channel, it has been demonstrated that the shorter 01 subunit is truncated at its COOH terminus compared to the longer isoform (De Jongh etal., 1989, 1991). T o examine the COOH-terminalregion of the two isoforms of the classC L-type calcium channel, we therefore produced the antibody CNC2 directed against the amino acid sequenceat the COOHterminal endof the classC L-type a1 subunit asdeduced from cDNA cloning and sequencing. In both immunoblotting and immunoprecipitation experiments, this antibody bound only to Lc2, the longer form of the class C a1 subunit, demonstrat-

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ing that Lc2 contains the COOH terminus encoded by the class C cDNA. These results also indicate that the COOH terminus of the shorter form, Lcl, is truncated in comparison to Lcz. In skeletalmuscle, the available evidence suggests that the two size forms of the a1 subunit arise by post-translational proteolytic processing because only a single mRNA has been characterized. In contrast,sequencing of cDNA clones encoding the neuronal P-type, N-type, and class D L-type calcium channels has revealed multiple isoforms in each case which are identical except for COOH-terminal regions of different length and are likely the result of alternative RNA splicing (Williams et al., 1992a, 1992b; Mori et al., 1991; Hui et al., 1991; Seino et al., 1992). Alternative splicing of a single rat class C gene has been shown to generate isoforms that differ in internal regions but have similar molecular sizes (Snutch et al., 1991). Thus, Lcl and Lcz may arise from further alternative splicing of a common mRNA transcript of the class C calcium channel gene, or LCl may be formed by specific proteolytic processing of LC2. Differential Phosphorylation of the Two Size Forms of the Class C L-type Calcium Channel-Although an increasing number of electrophysiological studies indicates that neurotransmitters and hormones modulate neuronal L-typecalcium channels by activating protein kinases, phosphorylation of neuronalL-typechannelshasnotyet been reported. Our results demonstrate direct phosphorylationof the class C Ltype a1 subunit by cA-PK, CaM kinase 11, cG-PK, andPKC. Lc2, the longer form of this subunit, isphosphorylated by all four kinases while Lcl is phosphorylated by PKC, cG-PK, and CaM kinase 11, but not by cA-PK. Thus, cA-PK differentially phosphorylates thetwo size forms of the a1 subunit. The most likely interpretation of this result is that cA-PK phosphorylates Lc2 within the COOH-terminaldomain which is not present inLcl. This is analogous to theskeletal muscle L-type channel inwhich the primary phosphorylation site of the longer a1 subunit isoform is in its COOH-terminal end, which is not present in the shorter form (Rotman etal., 1992). However, we cannot exclude the possibility that truncationof the class C a1 subunit at its COOH-terminal end causes a change in conformationwhich would prevent thephosphorylation of the shorterform by cA-PK.In eithercase, our results show that PKC, cG-PK, and CaM kinase I1 phosphorylate the class C a1 subunit at oneor more sites that aredifferent from the phosphorylation sitesfor cA-PK. The primary sequence of the rat neuronal class C L-type a1 subunit is 95% identical with that of the L-type calcium channel from rabbit heart, but most of the differences in amino acid sequence are concentrated in the COOH-terminal domain (Mikami et al., 1989; Snutch et al., 1991). Multiple lines of evidence indicate that the cardiac L-type channel is regulated by neurotransmitters and hormones by a cA-PKdependent mechanism (see Pelzer et al. (1990), for review). Nevertheless, attempts toshow direct phosphorylation of the cardiac calcium channel a1 subunit with an apparent molecular mass of about 210 kDa have been unsuccessful (Chang and Hosey, 1988; Yoshida et al., 1990). Recently, however, a second, minor form of the cardiaca1 subunit with amolecular mass of 250 kDa was detected with a novel antibody (Yoshida et al., 1992). Analogous to our findings, only the longer form was phosphorylated by cA-PKafter immunoprecipitation. Based on the results presentedhere, it may be expected that cA-PK phosphorylatesthe longer form of the cardiac a1 subunit at its COOH-terminal end and that thesmaller isoform is truncated at its COOH-terminal end. Physiological Relevance of the Phosphorylation of Neuronal

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L-type Calcium Channels-Electrophysiological data show that the activity of neuronalL-type calcium channels is regulated by neurotransmitters and neuromodulators through phosphorylation by cA-PK or PKC (see Dolphin, 1990, and Miller, 1990, for review). cA-PK increased neuronal calcium currents in GH3 pituitary cells (Armstrong and Eckert,1987), hippocampal neurons (Gray and Johnston, 1987), snail neurons (Doroshenko et al., 1984; Chad and Eckert, 1986), rat nodose ganglion cells (Gross et al., 1990), frog sympathetic ganglion neurons (Lipscombe et al., 1992), and chromaffin cells (Artalejo et al., 1990). PKC increased neuronal L-type calcium channel activity in frog sympathetic neurons (Yang and Tsien, 1993) but decreased it in cultured fetal hippocampal neurons (Doerner et al., 1990). The activity of neuronal calcium channels, which were not further identified, was also reduced by PKCincultured cerebral neurons (Werz and Macdonald, 1987), chicken embryonic dorsal root ganglion neurons(RaneandDunlap, 1986), and snail F1 neurons (Gershenfeld et al., 1991). In hippocampal pyramidal cells and most other neurons studied, L-type calcium channels are primarily located in the cell bodies and proximal dendrites (Ahlijanian et al., 1990; Westenbroek et al., 1990). This localization suggests that Ltype calcium channels are involved in general intracellular regulatory events including regulation of protein phosphorylation, gene expression, and cell morphology. It has been shown that calcium entry through L-type channels regulates gene expression in PC12 cells (Morgan and Curran, 1986),cultured cortical neurons (Murphy et al., 1991), and cultured fibroblasts (Block et al., 1991). In addition, neurite outgrowth in PC12 cells depends on the activation of L-type channels by cell adhesion molecules (Doherty et al., 1991). Modulation of the functional activity of the class C L-typecalcium channels by phosphorylation by cA-PK or PKC may have important effects on regulation of these biochemical processes. The existence of two or more a1 subunit size forms with variant COOH-terminal ends, as initially shown in skeletal muscle (De Jongh et al., 1989,1991), is emerging as a common feature of calcium channel diversity. Multiple size forms of calcium channel a1 subunits differing in their COOH-terminal regions have been observed for cardiac calcium channels (Yoshida et al., 1992), for class B N-type neuronal calcium channels (Westenbroek et al., 1992), and for both class C (this report) and class D3 L-type calcium channels. In this regard, it is noteworthy that all neuronal calcium channels cloned to date have been shown to possess unique COOH-terminal regions (Snutch and Reiner, 1992). Differential phosphorylation of these size forms by specific protein kinases creates the possibility for differential regulation. The in vitro phosphorylation experiments described here are a first step toward investigating the phosphorylation of the class C L-type channel in neuronal cells and its role in regulation of calcium channel function. Acknowledgments-We are grateful to Drs. Eric I. Rotman and Brian J. Murphy of this department for generously providing cA-PK and PKC, to Drs. D. A. Brickey and T. Soderling, Vollum Institute, for generously providing CaM kinase 11, to Dr. J. Corbin, Vanderbilt University, for generously providing cG-PK, and to Anita A. Colvin and Dr. Karen S. De Jongh, Molecular Pharmacology Facility, Department of Pharmacology, University of Washington, for synthesizing peptides. REFERENCES Ahlijanian, M. K., Westenbroek, R. E., and Catterall, W. A. (1990) Neuron 4 , 819-832

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